ThPOK plays a key role in thymic development, and potentially in mature T cell function. We propose studies to elucidate regulation of the ThPOK silencer, a key cis element that is necessary for lineage specific ThPOK expression, test the functional significance of Zfp281, a potential new regulator of ThPOK expression, and dissect the role of ThPOK in peripheral T cells, using a novel mouse strain (OB11 line) in which ThPOK is selectively turned off in peripheral CD4 T cells. Aim 1. Functional dissection of the ThPOK silencer. This aim has two objectives: 1) To test the novel hypothesis that NFAT and Egr factors control silencer function, by mapping functionally relevant NFAT and Egr consensus motifs, testing whether mutation of these sites affects Runx binding and epigenetic state of the silencer, and determining whether ectopic expression of NFAT and Egr factors can directly antagonize silencing. 2) To characterize a 100 bp regulatory motif that is required for ThPOK expression in mature CD4 T cells, and seems to mediate interchromosomal interactions. We will determine whether presence/absence of this motif affects the epigenetic state of the silencer, and use 3C and FISH approaches to address its potential role in nuclear repositioning of the ThPOK locus. Aim 2. Analysis of the role of Zfp281 in control of ThPOK transcription and T cell development/function. A Y1H screen revealed Zfp281 as a potential new regulator of ThPOK transcription. Multiple additional lines of evidence suggest a role for Zfp281 in ThPOK regulation, and T cell development/function. To test this directly, we will generate and fully characterize a conditional T cell-specific Zfp281 knockout mouse. Additionally, we will use Zfp281-GFP reporter mice to assess Zfp281 expression at the single-cell level during thymic development, and determine functional consequences of mutating Zfp281 consensus motifs within the ThPOK silencer and distal promoter elements. Defining Zfp281 as a novel regulator of T cell development/function would represent an important conceptual advance. Aim 3. Defining the role of ThPOK in peripheral T cell function. Mice that selectively lack ThPOK in peripheral CD4 T cells exhibit increased expression of IL-9, IL-17 and IL2R. We propose to elucidate the underlying mechanism for derepression of these genes and determine whether downmodulation of ThPOK is important for promoting cytokine expression during normal Th differentiation. First, we will investigate whether derepressed genes are direct targets of ThPOK regulation, and if so, whether loss of ThPOK results in epigenetic remodeling of target loci. Secondly, we will assess whether there is a selective requirement for ThPOK under different Th polarization conditions. Specifically, we will test whether the absence or constitutive expression of ThPOK preferentially affects certain Th polarization programs, and whether ThPOK expression is differentially regulated during Th polarization to particular lineages.